This grant proposal aims at investigating mechanisms of growth factor-induced cell cycle recruitment of leukemic cells, in vitro and in vivo and at developing optimized cytokine/chemotherapy regimens for patients with acute myeloblastic leukemia (AML). Rationale is based on the known cell cycle specificity of the main drugs used in the therapy of AML and MDS, i.e. ARA-C and anthracyclines, on prognostic models that recognize the negative impact of quiescent leukemic cells on therapeutic response and on the demonstrated recruitment of leukemic cells by granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3). We now propose to extend these studies to include the GM-CSF/IL-3 fusion molecule PIXY321, and the c-kit ligand stem cell factor (SCF). End points in these in vitro studies will be defined by multiparameter flow cytometry measurements of DNA, RNA, BUdR, IUdR and cell cycle-related proteins (c-myc, p53, PCNA) by results obtained in suspension cultures and in clonogenic assays. These studies of fresh leukemia samples (120 patients with newly diagnosed AML will provide detailed analysis of leukemia cell differentiation and proliferation, in particular of recruitment of Go cells into the cell cycle, and will identify the most active cytokine combinations. We will then expose the stimulated cells to ARA-C and to anthracyclines with the aim of maximizing cell kill by these agents. A newly developed FACS technique allows molecular analysis of gene expression in sorted hematopoietic cell populations of defined lineage and maturation, and will provide detailed analysis of lineage-specific gene regulation by cytokines. Fluorescence in-situ hybridization (FISH) using chromosome-specific probes will allow the crucial discrimination of cytokine effects on normal and leukemic cells. We already have evidence for selective cytokine effects on clonally abnormal leukemic cells, using the combined method of BUdR labelling and FISH analysis, and the proposed studies will allow us to optimize selective recruitment of leukemic cells. Clinical studies of IL-3 in AML will be hypothesis monitored by these new techniques and will allow us to test the that in vivo recruitment of hematopoietic cells is possible, and of therapeutic benefit.